Neural crest cell migration as a functional endpoint to test for developmental toxicity

Neurodevelopmental disorders affect 10-15% of children. For disorders like autism and attention deficit disorder, the prevalence has been rising, pointing towards environmental risk factors such as toxicants. At present, only few compounds are known as developmental neurotoxicants but most compounds have never been assessed for developmental neurotoxicity (DNT). To change this, novel high-throughput human cell-based assays are developed with the aim to model all critical steps of neurodevelopment.One step involves proper functioning of neural crest cells (NCC). NCCs are a particular fetal cell type that arises at the time of neurulation, delaminates from the neural tube and migrates to different places in the embryo to give rise to various cell types, including enteric and peripheral neurons, melanocytes and craniofacial skeleton. NCC function can be disturbed by toxicants such as retinoic acid.The aim of the present thesis was to develop a high-throughput assay to measure human NCC migration and to investigate the molecular mechanisms that are disturbed by NCC migration-inhibiting compounds.In a first step, an existing NCC migration assay was adapted for high-throughput. The new as- say (cMINC) is experimenter-independent, suitable for automated image acquisition and reproducible. Proliferation-inhibition by toxicants was identified as potential confounding factor and further investigated. Using appropriate positive and negative controls, a preliminary prediction model was set up.In a second step, performance of the assay was tested by screening a compound library consisting of compounds from several chemical classes (flame retardants, pesticides, drug-like compounds, polycyclic aromatic hydrocarbons, industrial chemicals), including known DNT compounds and negative controls. Of the tested compounds, 23/75 disturbed NCC migration at non-cytotoxic concentrations. Strikingly, many hits were halogenated or phosphorous organic compounds.In a third subproject, mechanisms of migration-inhibition were investigated using polychlorinated biphenyls (PCBs) as an example. PCBs are a group of environmental contaminants composed of 209 congeners with varying chemical properties and biological activities. A structure-activity-relationship (SAR) was built for migration-inhibition and compared to literature data and own measurements. Subsequently, reduction of Connexin43 (Cx43) plaque number was identified as a potential key event in migration-inhibition.In a follow-up project, testing of other NCC migration-inhibiting compounds revealed that several toxicants interfered with Cx43 localization, indicating that Cx43 mislocalization might be a general toxicity mechanism. Moreover, Cx43 is known to play an important role in NCC migration in vivo. All together, this leads to the conclusion that Cx43 mislocalization is a likely mechanistic step involved in migration-inhibition by a subset of NCC toxicants.To conclude, the developed migration assay is suitable for medium to high throughput. Over 100 compounds have already been tested and > 30 NCC migration-inhibiting compounds identified. Some hits are shared with other assays, but there are also compounds unique to the cMINC assay. This indicates that the assay could give complementary information as part of a test battery to assess DNT. Future studies should address the role of Cx43 localization in human NCC migration in more detail.publishe